结核分枝杆菌PPE61蛋白的表达与纯化及其对BEAS-2B细胞增殖和TGF-β表达的影响OA

PPE61,a member of the PPE protein family specific to Mycobacterium tuberculosis,plays a key role in immune evasion and host-pathogen interactions,as implicated by its variable C-terminal region and secretion properties.However,its specific functions in respiratory barrier epithelial cells remains unclear.In order to explore,through in vitro experiments,the regulatory role of PPE61 on the proliferation of human bronchial epithelial cells(BEAS-2B)and its molecular mechanisms during early infection,and to provide a theoretical basis for tuberculosis diagnosis and vaccine development,the fundamental biological properties of PPE61 protein were predicted using bioinformatics software,PPE61 gene and its encoded protein sequence information were obtained from NCBI database.PPE61 gene was amplified via PCR from Bacillus Calmette-Guérin(BCG)genome,and a recombinant expression plasmid,pET-22b(+)-PPE61,was constructed and transformed into Escherichia coli(E.col)BL21(DE3)for protein expression.Protein expression and antigenicity were verified by SDS-PAGE and Western blot;the proliferative activity of BEAS-2B cells treated with different concentrations(2.5-20 μg/mL)of PPE61 for 24 hours was detected using CCK-8 assay,and the level of TGF-β gene expression was analyzed by qPCR.The results showed that the amplified PPE61 gene was 1 287 bp in length,encoding 406 amino acids,the protein lacked a signal peptide and transmembrane regions,but was predicted to contain 37 phosphorylation sites,41 B-cell epitopes and 35 T-cell epitopes,and its secondary structure was predominantly alpha-helical(49.26％);SDS-PAGE analysis revealed that the protein was available in the form of inclusion body with an approximate molecular weight of 105.652 18×105,consistent with its expected size,indicating that target protein could be induced and expressed after the recombinant plasmid was transformed into competent E.coli BL21(DE3)cell.It was confirmed that,by Western blot,PPE61 antigen was specific;its dose-dependently inhibited cell proliferation(P≤0.000 1)and significantly upregulated TGF-βexpression(P＜0.05).In conclusion,PPE61 protein had rich antigenic epitopes and high solubility,indicating good immunogenicity and promising prospects for development,by which a new target was provided for screening markers of tuberculosis diagnosis and developing novel subunit vaccines,and an important experimental foundation was laid.